Ojective 1: Develop approaches for expansion of hematopoietic stem cells (HSCs) Attempts to improve hematopoietic reconstitution and engraftment potential of ex vivoexpanded hematopoietic stem and progenitor cells (HSPCs) have been largely unsuccessful due to the inability to generate sufficient stem cell numbers and to excessive differentiation of the starting cell population. Protocols that are based on hematopoietic cytokines (e.g. thrombopoietin TPO, stem cell factor SCF) have failed to support reliable amplification of immature stem cells in culture, suggesting that alternative cytokines or additional factors are required and highly desirable. ELTROMBOPAG. Eltrombopag is a novel TPO agonist with an intrinsic ability to expand HSPC in vivo given observed clinical benefits in patients with aplastic anemia. We have cultured human CD34+ cells for up to 21 days in our standard culture medium supplemented with Eltrombopag or with the combination Eltrombopag + TPO using various cellular or non-cellular support: 1) Fibronectin; 2) Osteoblasts; 3) Endothelial cells. We found that Eltrombopag was not superior to TPO for HSPC expansion in vitro in the absence of a cellular support. Results of transplantation of CD34+ cells expanded on osteoblasts or endothelial support into immune-deficient mice, the gold standard for evaluation of the in vivo repopulating potential of these cells, are pending and will determine the clinical utility of this drug for in vitro expansion of HSCs. Ongoing studies in our laboratory indicate that this drug may work by induction of DNA repair pathways with resulting decrease in apoptosis in HSPC. NOTCH PATHWAY AND HYPOXIA. The primary mediators of hypoxic adaptation are hypoxia-inducible factors (HIF), a family of transcription factors composed of two subunits, an oxygen-labile &#945; subunit that rapidly stabilizes in response to low O2 tensions (HIF-1&#945; and HIF-2&#945;), and a &#946; subunit (HIF-1&#946;) that is constitutively expressed. In immunoprecipitation assays, HIF-1&#945; physically interacted with the intracellular domain of Notch, a critical component for the maintenance of undifferentiated stem and progenitor cell populations, providing a striking molecular link between hypoxia and stemness. Given this recent evidence of a convergence of pathways involved in hypoxia sensing and stem cell maintenance, we are investigating the possibility of stem cell expansion under hypoxic conditions by activation of the canonical Notch signaling pathway using Delta-1 ligand. We have shown that culture of human HSPC under hypoxic conditions in the presence of Notch ligand resulted in 5-fold expansion of the most primitive hematopoietic cells with engraftment potential compared to cells cultured under hypoxia in the absence of Notch ligand. Additional confirmatory studies are ongoing and possible clinical applications are considered. NOVEL PATHWAYS FOR HSC EXPANSION. During homeostasis the HSC pool is maintained at a relatively constant level. In contrast, several murine studies have shown that during hematopoietic stress, such as transplantations, HSCs can and will self-renew extensively, suggesting that HSCs are exposed in vivo to specific factors/signals that promote their self-renewal and amplification. We have transplanted human CD34+ cells into immune-deficient mice and showed evidence of extensive self-renewal in vivo. To identify novel factors/pathways involved in HSC expansion, we are comparing gene expression/methylation patterns between HSC at steady state (before transplant) and after transplant, using RNA-Seq, CHIP-Seq and metabolomics approaches. Optimal transplantation conditions have been identified in the past year and comparative global transcriptome analyses are ongoing. These studies will have a significant impact on the global understanding of human HSC self-renewal and could lead to the development of novel approaches for HSC expansion in vitro. development of novel approaches for HSC expansion in vitro. Objective 2: Develop approaches for differentiation of iPSCs into HSCs With the development of induced pluripotent stem cell (iPSC) technologies emerged the concept of generating iPSCs from an individual patient, correcting the genetic defect using gene specific targeting for safe integration of the therapeutic transgenes, and differentiating the disease-free iPSCs into transplantable HSCs. Currently used protocols for iPSC differentiation into HSCs can generally be divided into two main categories: those that co-culture stem cells with stromal layers (e.g. OP9), and those that culture stem cells in suspension to form embryoid bodies (EBs). However, these protocols remain inefficient at producing the quantity and quality of HSCs required for clinical applications. On the basis of recent data demonstrating that definitive HSCs are generated from a unique population of endothelial cells known as hemogenic endothelium (HE), we have established a novel system for de novo generation of transplantable HSCs from iPSCs. Human iPSCs derived from normal individuals are cultured in the presence of a cytokine combination that favors development of an adherent layer of HE in vitro. Over a period of 12-14 days, these cells further differentiate to produce and release cells in suspension. Up to 70% of these cells have a CD45+CD34+ phenotype compared to 10-15% CD45+CD34+ using current co-culture or EB-based protocols. We have characterized these cells further and demonstrated a subpopulation with the most defined HSC phenotype described (CD34+CD38-CD45RA-CD90+CD49f+Rholo). We have demonstrated that these cells can generate hematopoietic progenitors in vitro but they are functionally inapt at establishing hematopoiesis in vivo, suggesting a developmental defect in iPSC-derived HSPC. Various approaches are underway to address this fundamental issue: 1) Comparative global transcriptional analysis of primary and iPSC-derived HSPC; 2) Instructive potential of endothelial cells; 3) Differentiation of iPSC under hypoxic conditions; 4) Optimization of currently used approach for mesodermal differentiation. We have also initiated the derivation and genetic correction of iPSC lines derived from patients with life-threatening bone marrow failure syndromes for eventual clinical applications. Objective 3: Initiate a first-in-human clinical trial of gene therapy for patients with leukocyte adhesion deficiency type 1 (LAD-1) Patients with Leukocyte Adhesion Deficiency Type 1 (LAD-1) suffer life-threatening bacterial infections that stem from the inability of their neutrophils to adhere to blood vessel walls and migrate to sites of infections. Heterogeneous genetic mutations in the leukocyte integrin CD18 subunit have been associated with this abnormal phenotype. HSPC transplantation represents the only curative therapy for LAD-1, but there are well-described limitations with this treatment. Gene therapy represents an alternative, potentially safer, curative option for these patients. Foamy Viral Vectors (FVV) have several advantages over currently used gene transfer vectors. In pre-clinical studies, we demonstrated correction of the LAD-1 phenotype in four dogs with canine leukocyte adhesion deficiency (CLAD), the animal counterpart to LAD-1 in humans. There has been no emergence of clonal dominance or leukemia after several years of follow-up. In this project, we are developing a human gene therapy clinical trial for LAD-1 using FVV. This project is conducted in two consecutive phases: 1) Development of methods for production, certification and testing of transduction efficiency of cGMP-like and cGMP grade lots of &#916;&#934;MSCV-hCD18 FVV for clinical use; 2) Patients with LAD-1 will be enrolled and monitored for safety and efficacy in a non-randomized phase I gene therapy clinical trial.